SUMMARY Once considered a death sentence, infection with HIV is now a manageable, chronic disease. To keep the viral load low and to prevent progression to AIDS, patients are required to adhere to a regime of multiple antivirals for life. Long-term treatment does not eliminate the virus and can lead to serious toxicities. Therapies that either cure infection or that offer improvements in convenience or tolerability are needed. Current anti-HIV therapeutics target fewer than half a dozen protein active sites. Significant recent advances in the structural biology of HIV offer an opportunity to develop therapeutics that target tertiary structures within the HIV RNA genome that are essential to the viral replication cycle. RNA is a compelling target for small-molecule drug discovery, and the genomes of RNA viruses, such as HIV, contain highly conserved targetable structures. Multiple natural products target ribosomal RNA, establishing proof of concept for RNA as a drug target; however, RNA-targeted drug discovery remains a nascent field. Specific tertiary structures in the HIV RNA genome are essential for viral replication, and we are ideally positioned to develop a screening system for small molecules that bind to these tertiary structure elements. The well-characterized Rev response element (RRE) forms a complex with Rev protein that is essential for export of HIV RNAs from the nucleus, a process necessary for productive infection. We will employ the Ribometrix lead discovery platform to identify small molecules with favorable medicinal chemistry properties that bind to the RRE to block binding of Rev or to disrupt the tertiary structure recognized by Rev. These leads will serve as a foundation for development of novel anti-HIV therapeutics. At Ribometrix, our lead discovery platform combines SHAPE-MaP, a high-quality, rigorous biophysical technology for RNA structure analysis, and fragment-based drug discovery (FBDD), a well-validated strategy for lead compound identification that allows efficient sampling of chemical space. The strategy we propose to develop here can be used in efforts to identify small molecules that bind to and disrupt structures of diverse therapeutically important RNAs. Thus, we are poised to fully test and establish proof-of-principle for an efficient and generic approach for RNA-targeted ligand discovery, focused on a critical step of the HIV replication cycle.